Protistology 4 (1), 7378 (2005) Protistology Response of tolerant and wild type strains of Chlorella vulgaris to copper with special references to copper uptake system A. A. Fathi 1, F. T. Zaki 2 and H. A. Ibraheim1 1 Botany Department, Faculty of Science, ElMinia University, ElMinia, Egypt 2 Botany Department, Faculty of Science, Cairo University, Giza, Egypt Summary Copper tolerance in Chlorella vulgaris has been studied by comparing physiological properties and copper uptake in a wild type strain and a copper tolerant one. A concentrationdependent reduction in growth rate, dry mass, and content of chlorophyll a, protein, sugars and amino acids was noticed in both strains at 1.0 and 400 !g l1 copper. The reduction in all parameters was higher in the wild type strain than in the tolerant one. As compared to the wild strain, the tolerant strain showed insignificant loss of potassium and sodium. The data on proline accumulation showed a positive relationship between copper toxicity and proline accumulation in both strains, more pronounced in the tolerant strain. The copper uptake was influenced by the copper concentrations the algae had been exposed to during their previous growth: the lower the copper concentration in the culture medium, the higher the activity of the uptake and the capacity of the cells to accumulate copper. Key words: Chlorella vulgaris, copper, tolerance, uptake Introduction Fathi and ElShahed, 2000; Fathi, 2002; Backor et al., 2003; Bossuyt and Janssen, 2004). Tolerance to heavy metals in different organisms, Copper is an essential trace element for plants, including algae, may develop either during growth in because it participates in photosynthetic electron metalcontaminated natural habitats or through transport and plays a role as cofactor of several oxidizing successive cultivation at elevated doses of heavy metals enzymes. However, similarly to many trace elements, in the laboratory (Rai et al., 1991). Cases of acquired copper is toxic at higher concentrations, resulting in resistance to copper in algae growing in Cucontami an inhibition of electron transport (Shioi et al., 1978), nated freshwater habitats are numerous (Rai et al., 1991; inhibition of photosynthetic pigments (Fathi et al., Fathi and Falkner, 1997; Lombardi and Vieira 1998; 2000), and decrease in intracellular potassium and © 2005 by Russia, Protistology 74 · A. A. Fathi, F. T. Zaki and H. A. Ibraheim sodium concentrations (De Filippis, 1979). A lack of A standard initial inoculum of the wild type and copper, on the other hand, can partially be compensated, the tolerant strain was inoculated to culture flasks (250 e.g. by substitution of phycocyanin by cytochrome c ml each) that contained 50 ml of nutrient medium (Sandmann and Boger, 1980). supplied with 1.0 and 400 !g l1 copper and basal In the present paper we investigate copper tolerance medium as a control. At the end of incubation period in the green alga Chlorella vulgaris (isolated from the (7 d), cultures were filtered and washed several time River Nile) by comparing a wild type and a tolerant with copper free medium for different measurements. strain exposed to different copper concentrations with At least three replicates for each sample and control special references to their copper uptake systems. were used. The number of cells was determined using a Material and methods Haematocytometer chamber. Pigments were estimated in acetone extract according to Metzner et al. (1965). Chlorella vulgaris Beyerinck was isolated from the Sugar content was determined by the anthrone method River Nile at ElMinia (Egypt). The alga was grown in (Roe, 1955), using glucose as a standard. Total amino Kuhl's medium (Kuhl 1962), modified as follows: copper acid content was determined according to Moore and was eliminated, as were all known chelators (ferric citrate, Stein (1948). Total protein was measured according to citric acid, and NaEDTA), and trace metal levels were Lowry et al. (1951). Proline content of cells was reduced to 1/20th of the original amount proposed by estimated in aqueous extract using the acid ninhydrin Kuhl. The concentration of copper (provided to the method described by Bates et al. (1975). The K+ and medium as copper sulfate) was 0.5 !g l1 (as in the river Na+ present inside the cells were released by heating water). In order to simulate the natural situation, when the washed pellets in boiling water bath. Cells were then phosphate needed for growth is not always available and removed by centrifugation and the amounts of K+ and therefore algae change between phosphate deficient and Na+ were determined with the help of a Clinical flame nondeficient states, cultivation was performed in a photometer Corning 41# C (Corning Science Products, discontinuous mode using 200 ml medium that con Halstead, England) according to Rai et al. (1991). tained 10 !M phosphate. Every 24 hours, 100 ml of the Copper uptake by algal cells was measured algal cultures were removed and replenished by fresh according to Fathi and Falkner (1997) by a Perkin medium again containing 10 !M phosphate. After Elmer Atomic Absorption Spectrometer with graphite dilution, phosphate was rapidly incorporated by the furnace (Model 4000, Norwalk, USA). The amount remaining algae and stored in form of polyphosphates, incorporated was calculated from the decrease of the which served as an endogenous phosphorous source for copper concentration in the incubation medium, growth. Cultures were grown at 27 @ 1.0# C at a 16 h divided by the cell number and expressed in attograms light: 8 h dark cycle with a light intensity 70! mol m2s1. Cell1 = 1018 g cell1. The values calculated were the Seven days cultures (optimal growth period) were diluted mean of triplicates, the standard deviation was less than under aseptic conditions to provide stock cultures. All 5% of these mean values. The copper content of the solutions were prepared using Millipore membrane filter algae was determined after digestion of the washed and ! z 0.45 M (Schleicher and Sch ll, Germany). All glassware dried material for 15 min in a boiling mixture of HNO3 was soaked for 24h in 1015% nitric acid, rinsed in and HCl (1:1 v/v). distilled water, and airdried before use. A copper tolerant strain of Chlorella vulgaris was Results and Discussion isolated by successive culturing on agar plates (Kuhl's medium) containing different doses of copper (0.05 to The data in Table 1 show that copper supple 0.5 mg dm3), as described by Whitton and Shehata mentation severely reduced growth rate, dry mass, (1982). Colonies appearing on plates containing 0.4mg chlorophyll a, protein, sugars and amino acids content dm3 copper were picked up and transferred repeatedly of the nontolerant strain of Chlorella vulgaris in a to agar plates with the same concentration of copper. concentrationdependent manner. However, the impact Colonies growing successfully on such plates and also was insignificant in the tolerant strain. A concentration in liquid medium containing 0.4 mg l1 copper were dependent reduction in the parameters investigated designated as the copper tolerant strain. When this (Table 1) is in good agreement with the previous findings tolerant strain was subcultured in the basal medium (Fisher and Frood, 1980; Sato et al., 1986; Rai et al., devoid of copper and then transferred to the medium 1991; Fathi and ElShahed, 2000). with 0.4 mg l1 copper, a gradual loss of tolerance in The results of this investigation clearly show that the strain was noticed after every successive generation increased metal concentration depresses the photo (Rai et al., 1991). Hence, the strain isolated was not a synthetic activity of the two strains of Chlorella vulgaris, spontaneous mutant but a physiologically adapted one. causing lower Chla content especially in the non Protistology · 75 Table 1. Effect of 1.0 and 400µg/l copper on growth rate, dry mass, the content of Chla, amino acids, protein, sugars and proline , as well as the potassium and sodium contents in the wild type and the coppertolerant strain of Chlorella vulgaris measured after 7 days treatment. Mean of three replicates + SE. Data in parentheses denote percentage of inhibition. Strain Nontolerant Tolerant Copper conc. (µg/l) Cont. 1.0 400 Cont. 1.0 400 Growth rate (division /d) 0.51 + 0.01 0.25 + 0.00 0.02 + 0.00 0.65 + 0.00 0.57 + 0.01 0.55 + 0.02 (51.00) (96.08) (12.31) (15.38) Dry mass (mg l1 42.61 + 0.10 20.51 + 0.20 6.32 + 0.01 45.33 + 0.20 38.64 + 0.10 38.00 + 0.20 medium) (52.00) (85.20) (14.75) (16.17) Chla ( µg/ mg dry mass) 2.47 + 0.02 1.66 + 0.02 0.20 + 0.00 5.56 + 0.05 5.50 + 0.04 5.50 + 0.04 (32.80) (91.90) (1.08) (1.08) Amino acids (% dry 34.31 + 0.04 25.00 + 0.01 10.61 + 0.01 38.00 + 0.02 36.82 + 0.02 35.56 + 0.02 mass) (27.13) (69.07) (3.10) (6.42) Protein (% dry mass) 31.42 + 0.10 15.33 + 0.20 5.21 + 0.01 31.02 + 0.20 28.60 + 0.02 27.75 + 0.10 (51.21) (83.42) (7.80) (10.54) Sugars (% dry mass) 80.53 + 0.05 33.00 + 0.02 12.60 + 0.01 82.20 + 0.25 78.71 + 0.02 78.06 + 0.20 (52.81) (84.35) (4.24) (5.04) K+ (g/g protein) 0.36 + 0.03 0.12 + 0.00 0.02 + 0.00 0.39 + 0.00 0.35 + 0.00 0.36 + 0.00 (66.70) (94.45) (10.25) (7.69) Na+ (g/g protein) 0.25 + 0.01 0.05 + 0.00 0.02 + 0.00 0.28 + 0.00 0.25 + 0.00 0.25 + 0.20 (80.00) (92.00) (10.71) (10.71) Proline (x 108 ng/cell) 6.61 + 0.02 8.22 + 0.20 10.00 + 0.02 21.53 + 0.02 28.00 + 0.02 30.12 + 0.01 tolerant strain.